Are sperm parameters able to predict the success of assisted reproductive technology? A retrospective analysis of over 22,000 assisted reproductive technology cycles

Abstract Background An explosive increase in couples attending assisted reproductive technology has been recently observed, despite an overall success rate of about 20%–30%. Considering the assisted reproductive technology‐related economic and psycho‐social costs, the improvement of these percentages is extremely relevant. However, in the identification of predictive markers of assisted reproductive technology success, male parameters are largely underestimated so far. Study design Retrospective, observational study. Objectives To evaluate whether conventional semen parameters could predict assisted reproductive technology success. Materials and methods All couples attending a single third‐level fertility center from 1992 to 2020 were retrospectively enrolled, collecting all semen and assisted reproductive technology parameters of fresh cycles. Fertilization rate was the primary end‐point, representing a parameter immediately dependent on male contribution. Pregnancy and live birth rates were considered in relation to semen variables. Statistical analyses were performed using the parameters obtained according to the World Health Organization manual editions used for semen analysis. Results Note that, 22,013 in vitro fertilization and intracytoplasmic sperm injection cycles were considered. Overall, fertilization rate was significantly lower in patients with abnormal semen parameters compared to normozoospermic men, irrespective of the World Health Organization manual edition. In the in vitro fertilization setting, both progressive motility (p = 0.012) and motility after capacitation (p = 0.002) significantly predicted the fertilization rate (statistical accuracy = 71.1%). Sperm motilities also predicted pregnancy (p < 0.001) and live birth (p = 0.001) rates. In intracytoplasmic sperm injection cycles, sperm morphology predicted fertilization rate (p = 0.001, statistical accuracy = 90.3%). Sperm morphology significantly predicted both pregnancy (p < 0.001) and live birth (p < 0.001) rates and a cut‐off of 5.5% was identified as a threshold to predict clinical pregnancy (area under the curve = 0.811, p < 0.001). Discussion Interestingly, sperm motility plays a role in predicting in vitro fertilization success, while sperm morphology is the relevant parameter in intracytoplasmic sperm injection cycles. These parameters may be considered reliable tools to measure the male role on ART outcomes, potentially impacting the clinical management of infertile couples.


INTRODUCTION
The advent of assisted reproductive technology (ART) represented one of the most relevant medical innovations of the last century, drastically changing reproductive medicine. Different techniques and several treatment protocols evolved since the ART debut in 1978, but the vast majority of research studies in this setting remain focused on the same question, that is, how to increase ART success. To this aim, the identification of reliable and accurate predictors of ART outcome is mandatory. Since the use of assisted fertilization is accompanied by considerable economic and psycho-social costs, 1 the detection of predictive markers would be very useful to choose the ART type. However, the vast majority of studies evaluated only the female partner, whereas the male counterpart was relegated to a secondary role.
The studies considering common sperm parameters obtained by conventional semen analysis, that is, sperm number, motility, and morphology, 2 generated important threshold values that are commonly applied in clinical practice. Considering the less artificial ART approach, that is, intrauterine insemination (IUI), the total number of progressively motile sperm detected in fresh ejaculate resulted not predictive in terms of pregnancy achievement. 3,4 However, the number of inseminated, progressively motile spermatozoa was detected as a reliable predictor of pregnancy, 5 using one million as the clinical decision threshold to discriminate whether to send the couple to in vitro fertilization (IVF) or to IUI procedures. 4,6 However, infertile couples are more often addressed to more invasive techniques, such as IVF, irrespective of sperm quality-related parameters. In the IVF setting, in which the embryo is obtained placing the spermatozoa and the oocyte in the same culture medium, 7 the dependency of the outcome from sperm motility is expected. 8 Accordingly, sperm motility thresholds are recommended to opt for IVF, that is, total sperm motility higher than 30% and progressive motility higher than 15%. 9 Although it is reasonable to assume that sperm kinematic parameters could be relevant to improve IVF success rate, the current literature shows conflicting results, reporting a direct correlation between sperm progressive motility and pregnancy rate in some cohorts, 8,10 but not in others. 11 Another sperm quality-related parameter investigated in this setting is sperm morphology. However, the probability of IVF fertilization suc-cess seems to be independent of the percentage of normal forms, excluding its role as a reliable prognostic marker. 12 Since the advent of the intracytoplasmic sperm injection (ICSI), the use of conventional IVF progressively decreased. 13 Despite the direct microinjection of a single spermatozoon into the oocyte, the use of non-motile spermatozoa seemed to negatively impact fertilization outcome. 5 In addition, similarly to IVF, the prognostic value of sperm morphology remains poor also in the ICSI context. 12 Indeed, a meta-analytic approach failed to detect any association between isolated teratozoospermia and pregnancy rate, independently from the ART procedure used. 14 The potential contribution of the male gamete to the final ART outcome has been evaluated using innovative indicators of sperm quality, such as sperm DNA fragmentation index (DFI). 15 It has been hypothesized that an increased sperm DNA damage, exceeding 15%, could induce apoptotic pathways' activation, promoting embryo arrest. 16,17 However, conflicting results emerged even about DFI, since meta-analyses reported both a reduction 18 and no difference 19 in pregnancy rates in cases of high DFI levels in IVF and ICSI cycles.
Obviously, no convincing evidence that ART outcomes may be dependent on sperm parameters exists. Moreover, the variation over the years of the reference values in semen analysis, together with the poor inter-laboratory standardization of sperm assessment, contributes to this very complex scenario. 20 It remains undeniable that identifying thresholds of seminal parameters with prognostic significance in terms of pregnancy rate would have a considerable clinical impact in the assisted reproduction field. In this study, we aimed to answer the question of whether and which semen parameters can be useful for a priori prediction of ART outcomes. For this purpose, a large retrospective cohort analysis was conducted in a single third-level fertility center.

Study design and participants
A single-center, retrospective, observational study was carried out. All couples attending the Santa Maria Nuova Hospital of Reggio Emilia (Reggio Emilia, Italy) for primary or secondary infertility between January 1992 and December 2020 were considered eligible. During the time-frame interval evaluated, two historical moments (2005-2008 and 2018-2020) should be carefully considered, since structural interventions in the center and the COVID-19 pandemic reduced the number of fresh ART cycles performed.
Couple infertility was defined as the absence of conception after 12 months of unprotected sexual intercourse. 21 Accordingly, the transfer was performed at the blastocyst stage, with a maximum number allowed of one embryo for women younger and two for women older than 38 years. Semen sample preparation was performed as described elsewhere. 23,24 Briefly, an appropriate aliquot of fresh semen was diluted with 10 ml of buffered medium. After centrifugation (10 min at 800 x g at room temperature), the supernatant was removed and replaced by 5 ml of buffered medium. After a second centrifugation, the supernatant was removed once again, and the pellet was overlaid with 1 ml of medium and incubated (37 • C, 6% CO 2 in air) to separate motile spermatozoa by swim-up. After liquefaction, the sample was concentrated by one centrifugation (1500 x g), and the pellet was

Outcomes
The main outcome was fertilization rate, considered as the ratio between the number of fertilized oocytes and the number of injected/inseminated oocytes. This parameter was selected as the primary end-point since it represents the earliest outcome in which the male contribution can be assessed. Thus, this parameter was selected to verify the male role in ART processes, using earlier parameters, compared to strong ART outcomes. Secondary endpoints were the strongest ART outcomes, that is, pregnancy and live birth rates. The pregnancy rate was considered both as biochemical and clinical pregnancy. The former was defined by the detection of high levels of hCG in serum or urine, the latter was diagnosed by ultrasonography visualization or clinical documentation of at least one fetus with a heartbeat (including ectopic pregnancies) expressed for 100 initiated cycles, aspiration cycles, or embryo transfer cycles. Moreover, semen parameters were collected as specified in the following paragraph.

Semen analysis
Semen analyses were performed according to the World Health Organization (WHO) manual available at the time of the ART cycle. All semen analyses were performed by the same laboratory, undergoing regular internal and external quality controls over the years, and awarded the European Society of Human Reproduction and Embryology quality certificate. 25 Semen parameters were analyzed for volume, sperm count, sperm motility, and sperm morphology. Considering the wide time-frame interval of our study, three versions of the WHO manual were consecutively used during the data collection. [26][27][28] First, semen analyses have been performed using the 3rd edition, 26 whereas in 1999, the 4th edition 27 and in 2010, the 5th edition 28 were introduced. Each WHO manual edition introduced methodological differences and changed reference ranges. Since the 1992 and 1999 WHO manuals showed only minor changes in both methodologies and reference ranges, the results obtained using the two editions were considered together. In particular, oligozoospermia was defined by sperm concentration <20 million/ml and/or total sperm count <40 million, asthenozoospermia by progressive motility <25% and/or total motility < 50%, and teratozoospermia by sperm morphology <30% (3rd edition) 26 or 14% (4th edition). 27 On the contrary, the 5th edition defined oligozoospermia for sperm concentration <15 million/ml and/or total sperm count <39 million, asthenozoospermia for progressive motility <32% and/or total motility <40%, and teratozoospermia for sperm morphology <4%. 28 The capacitation test was routinely performed for all patients in each semen sample in order to select the proper ART methodologies to be applied. Capacitated spermatozoa were obtained by first selecting motile spermatozoa by a swim-up technique and then incubating the motile gametes in the Biggers-Whitten-Whittingham medium containing 35 mg/ml human serum albumin for 6 h. [29][30][31] The swim-up technique used for capacitation was the same preparation method as was used to prepare spermatozoa.

Statistical analysis
The entire cohort was first analyzed by descriptive analyses, considering both the female and the male partners. Semen analysis parameters were considered according to the WHO manual used for the interpretation and the sperm alterations classification was compared considering the cut-off suggested by the WHO manual used, as reported above.
A descriptive analysis of strong ART outcomes, that is, pregnancy and live birth rates, was provided for the entire cohort of cycles.

RESULTS
Twenty-two thousand and thirteen fresh ART cycles were performed between January 1992 and December 2020. Among these, 1545 (7.0%) cycles involved couples with secondary infertility (

IVF cycles
The fertilization rate was compared among classes of semen abnormalities, defined by the WHO manual's 5th edition and previous editions, separately (Table S1). Considering the WHO manual's 5th edition, fertilization rate was significantly lower in oligozoospermic patients (p = 0.030), in asthenozoospermic (p < 0.001), in teratozoospermic

F I G U R E 1 Box plots showing correlation between fertilization rate and sperm concentration (panel A and D), progressive motility (panel B and E), and sperm morphology (panel C and F). Redline shows the correlation trend
(p < 0.001), and in oligo-astheno-teratozoospermic (OAT) (p < 0.001), compared to normozoospermic patients (Table S1). Similar results were obtained using previous editions, showing lower fertilization rate in oligozoospermia (p < 0.001), asthenozoospermia (p < 0.001), teratozoospermia (p < 0.001), and OAT (p < 0.001), compared to normozoospermic patients (Table S1). As a confirmation, fertilization rate was directly correlated with sperm concentration (5th edition:    Table S2 shows the average sperm motility and morphology dividing couples who achieved and who did not achieve pregnancy.

ICSI cycles
The fertilization rate after ICSI was significantly lower in oligozoospermic (p < 0.001), asthenozoospermic (p < 0.001), teratozoospermic (p = 0.003), and OAT (p < 0.001), compared to normozoospermic patients using the WHO manual's 5th edition (Table S1). This difference was confirmed using parameters obtained according to previous WHO manual editions, with lower fertilization rate in oligozoospermia (p < 0.001), asthenozoospermia (p < 0.001), teratozoospermia (p < 0.001), and OAT patients (p < 0.001), compared to normozoospermic subjects (Table S1). As detected in IVF, ICSI-derived fertilization rate was directly correlated with sperm concentration  Finally, applying logistic regression analyses to strong ART outcomes prediction, sperm morphology significantly predicted both pregnancy (OR: 1.020, 95% CI: 1.008-1.032, p < 0.001) and live birth rates (OR: 1.018, 95% CI: 1.013-1.023, p < 0.001). These results confirm the potential predictive role of sperm morphology for the final ICSI outcome. Therefore, we performed ROC analyses using sperm morphology as a test variable and pregnancy and live birth rates as independent state variables. The first ROC analysis showed that a sperm morphology cut-off of 5.5% could be selected to predict clinical pregnancy (AUC = 0.811, standard error = 0.009, p < 0.001) with a sensitivity of 72% and specificity of 71% (Figure 2).
On the contrary, the ROC analysis performed using live birth rate as a state variable was not able to detect a sperm morphology threshold (AUC = 0.514, standard error = 0.024, p = 0.550). Table S2 shows the F I G U R E 2 Receiving operating characteristic (ROC) analysis using clinical pregnancy rate as a state variable and sperm morphology as test variable in intracytoplasmic sperm injection cycles average sperm motility and morphology dividing couples who achieved and who did not achieve pregnancy.

DISCUSSION
In our large cohort of fresh cycles, we confirm a higher fertilization rate in ICSI compared to IVF, as demonstrated in a recent meta-analysis. 34 This parameter could be considered the earliest and most suited variable to evaluate the male contribution to oocyte activation. 35 Indeed, fertilization rate, defined as the ratio between fertilized oocytes and injected/inseminated oocytes, is an informative, early parameter of the interaction of sperm plasma membrane and acrosome region with the oocyte within the sperm-cumulus oophorus complex, leading to egg activation. 36 While conventional IVF relies on co-incubation of oocytes with spermatozoa, ICSI consists of the injection of a single spermatozoon into the oocyte cytoplasm. 39 For this difference, ICSI was initially employed only for the treatment of infertility due to a severe male factor. However, nowadays it is largely preferred in clinical practice, up to 65% of the cycles, irrespective of semen quality. 40,41 This high and perhaps excessive use of ICSI is reflected by its routine use in many ART programs, and its preferential application in case of unexplained couple infertility and/or isolated low morphology on semen analysis. 42 However, despite ICSI representing one of the greatest advances in the field of assisted reproduction, bestowing men with severe oligozoospermia the opportunity to produce genetically own offspring, it does not seem to confer a concrete advantage over IVF in non-malefactor infertility. 43,44 Indeed, several studies suggested a higher risk of chromosomal abnormalities, 45  Different from ICSI, better IVF results, in terms of fertilization and pregnancy rates, are achieved in those couples in whom the male partner shows the highest sperm count and motility. Thus, we could speculate that IVF should be reserved for those cycles with normal sperm count and progressive motility, rather than sperm morphology. In this setting, similar results are obtained using motility after capacitation, rather than progressive motility alone. Thus, this parameter seems to be useful to help the clinician to select the appropriate ART cycle to apply in a given infertile couple. 68 Indeed, sperm capacitation, resulting in hyperactivated motility and acrosome reaction, naturally occurs for fertilization. 69 It is well known that despite normal semen parameters, some semen samples fail to fertilize the oocyte since the sperm is not able to capacitate. 69 With this in mind, it is clear that the evaluation of sperm motility after capacitation before IVF could be essential to better select the patient candidate for this ART technique. However, the final reproductive outcome, that is childbirth, seems to be unaffected by sperm motility. This result suggests that many other factors interfere with IVF success, other than sperm parameters. Thus, changing the point of view, the use of live birth rate as the outcome to evaluate male fertility-related treatment is incorrect and too far from male contribution.
Our results must be considered cautiously since the Italian law changing over time significantly impacted the efficacy of ART approaches. Indeed, the interpretation of the Constitutional Court of law number 40 in 2009 introduced important limits to ART, limiting the number of embryos transferred and contributing to increasing the use of cryopreserved oocytes. This change reduced ART efficacy in couples in which the woman's age was higher than 38 years and in couples in which the testicular sperm extraction was required. [70][71][72] However, in our cohort, we evaluated only fresh cycles and the mean female age was below this threshold, reducing this potential bias on our results. However, this selection reduced the sample size, probably reducing the final overall pregnancy rate obtained in the center. Moreover, this result should be due also to the long period of data collection, including two historical moments (2005-2008 and 2018-2020), in which structural interventions in the center and the COVID-19 pandemic reduced the number of fresh ART cycles performed. Moreover, our results, although statistically significant, show confidence intervals at the limits of significance. Therefore, this data must be carefully considered. Indeed, from entering the ART path to its outcome, the variables encountered are numerous. However, the identification of a single parameter that influences, albeit slightly, the final outcome, indicates a considerable clinical weight.
In conclusion, our results, derived from a large and homogenous cohort followed in a center, confirm the clinical relevance of semen parameters in the ART setting. In particular, sperm motility seems to be the best male parameter to predict fertilization rate in IVF. On the contrary, sperm morphology shows the strongest relationship in ICSI cycles, considering either fertilization rate or final ART outcomes, such as pregnancy rate. This result could have an immediate transposition into clinical practice, helping the choice of the best ART approach to be performed on the infertile couple or suggesting therapy for the male partner to pursue an improvement in sperm motility/morphology.